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目的构建含有人B7H4基因的重组逆转录病毒载体,获得稳定表达B7H4基因的L929细胞并初步研究其对T细胞的共信号作用及可能机制。方法从含人B7H4基因的cDNA序列FLJ22418中,采用PCR扩增出B7H4全长基因,双酶切装入逆转录病毒载体pEGZHATerm,与辅助病毒载体脂质体法共转染包装细胞293T,用其培养上清感染L929细胞72h后,经Zeocin筛选出稳定表达B7H4蛋白的L929细胞株;采用免疫荧光标记和流式细胞仪分析免疫分子的表达、3HTdR掺入检测细胞增殖以及ELISA测定细胞因子的水平。结果构建了含人B7H4基因的重组逆转录病毒载体和获得含有B7H4基因的重组病毒,筛选获得能稳定高表达人B7H4蛋白的L929转基因细胞;该转基因细胞对T细胞体外具有抑制增殖、活化和细胞因子分泌的作用。结论构建了稳定表达人B7H4蛋白的细胞株,B7H4分子在体外通过抑制T细胞分泌IL2和促进其凋亡作用可显著地下调T细胞功能的效应。
Objective To construct a recombinant retroviral vector containing human B7H4 gene and obtain L929 cells stably expressing B7H4 gene and to study its co-signaling role on T cells and its possible mechanism. Methods The full length B7H4 gene was amplified by PCR from the cDNA sequence FLJ22418 containing human B7H4 gene. The full length B7H4 gene was inserted into retroviral vector pEGZHATerm and co-transfected into 293T cells. After cultured in supernatant for 72h, L929 cells stably expressing B7H4 protein were screened by Zeocin. The expression of immune molecules was analyzed by immunofluorescence staining and flow cytometry. 3HTdR incorporation was used to detect cell proliferation and ELISA to determine the level of cytokines . Results Recombinant retroviral vector containing human B7H4 gene and recombinant virus containing B7H4 gene were constructed, and L929 transgenic cells stably expressing human B7H4 protein were screened out. The transgenic cells could inhibit the proliferation, activation and proliferation of T cells in vitro The role of factor secretion. Conclusion The cell line stably expressing human B7H4 protein was constructed. B7H4 could down-regulate the function of T cells by inhibiting the secretion of IL2 and promoting the apoptosis of T cells in vitro.